Published OnlineFirst July 3, 2018; DOI: 10.1158/1078-0432.CCR-18-0873 Translational Cancer Mechanisms and Therapy Clinical Cancer Research Mast Cell Activation and KSHV Infection in Kaposi Sarcoma Leona W. Ayers1, Arturo Barbachano-Guerrero2, Shane C. McAllister3, Julie A. Ritchie2, Elizabeth Asiago-Reddy4, Linda C. Bartlett4, Ethel Cesarman5, Dongliang Wang6, Rosemary Rochford2, Jeffrey N. Martin7, and Christine A. King2 Abstract Purpose: Kaposi sarcoma (KS) is a vascular tumor evaluated for levels of MC granule contents tryptase and initiated by infection of endothelial cells (ECs) with KS–as- histamine. sociated herpesvirus (KSHV). KS is dependent on sustained Results: In culture, MCs supported latent and lytic KSHV proinflammatory signals provided by intralesional leuko- infection, and infection-induced MC degranulation. Within cytes and continued infection of new ECs. However, the KS lesions, MCs were closely associated with spindle cells. sources of these cytokines and infectious virus within Furthermore, MC activation was extensive within patients with lesions are not fully understood. Here, mast cells (MCs) KS, reflected by elevated circulating levels of tryptase and a are identified as proinflammatory cells within KS lesions histamine metabolite. One patient with clinical signs of exten- that are permissive for, and activated by, infection with sive MC activation was treated with antagonists of MC proin- KSHV. flammatory mediators, which resulted in a rapid and durable Experimental Design: Three validated MC lines were used regression of AIDS-KS lesions. to assess permissivity of MCs to infection with KSHV and to Conclusions: Using complimentary in vitro and in vivo evaluate MCs activation following infection. Biopsies from 31 studies we identify MCs as a potential long-lived reservoir for AIDS-KS cases and 11 AIDS controls were evaluated by IHC for KSHV and a source of proinflammatory mediators within the the presence of MCs in KS lesions and assessment of MC KS lesional microenvironment. In addition, we identify MC activation state and infection with KSHV. Plasma samples antagonists as a promising novel therapeutic approach for KS. from 26 AIDS-KS, 13 classic KS, and 13 healthy adults were Clin Cancer Res; 24(20); 5085–97. Ó2018 AACR. Introduction occurring in immunosuppressed persons undergoing trans- plantation; and epidemic KS, occurring in patients with HIV/ Kaposi sarcoma (KS) is a unique, highly inflammatory AIDS. Independent of form, histologically, lesions are charac- "hemorrhagic sarcoma" initiated by infection of endothelial terized by proliferating spindle-shaped ECs and infiltrating cells (ECs) by KS–associated herpesvirus (KSHV; also known as leukocytes (1). Spindle cells carry latent KSHV, defined by human herpesvirus 8). There are four epidemiologic types of limited viral gene expression and no progeny production, but KS: Classic KS, which typically occurs in elderly men in Med- cells explanted from KS lesions and grown in culture rapidly iterranean regions and is relatively indolent; endemic KS, lose the KS genome (2). Thus, a source of infectious KSHV occurring in persons in sub-Saharan Africa; iatrogenic KS, virions within lesions capable of infecting new ECs is thought to be essential. Furthermore, while latently infected ECs secrete some inflammatory mediators, the levels are insufficient to 1Department of Pathology, The Ohio State University, Columbus, Ohio. 2Depart- ment of Microbiology and Immunology, SUNY Upstate Medical University, drive survival and growth of spindle cells in culture. Thus, fl Syracuse, New York. 3Department of Pediatrics, University of Minnesota Medical paracrine in ammatory signaling is thought to be essential for School, Minneapolis, Minnesota. 4Department of Medicine, SUNY Upstate Med- KS lesion development. The interplay between infection and ical University, Syracuse, New York. 5Department of Pathology and Laboratory inflammation allows for the enhanced survival of spindle cells 6 Medicine, Weill Cornell Medical College, New York, New York. Department of even prior to malignant transformation. Public Health and Preventative Medicine, SUNY Upstate Medical University, 7 Previous reports have described mast cells (MCs) in KS Syracuse, New York. Department of Epidemiology and Biostatistics, University fi of California, San Francisco, California. lesions but their signi cance has not been established (3). MCs are long-lived, tissue-resident cells that, like ECs, develop from þ Note: Supplementary data for this article are available at Clinical Cancer bone marrow–derived CD34 progenitor cells (4). Consistent Research Online (http://clincancerres.aacrjournals.org/). with their role in sensing pathogens and allergens, MCs are most Current address for R. Rochford: Department of Immunology and Microbiology, prevalent in mucosal tissues where they interface directly with University of Colorado, Denver, Aurora, Colorado. the external environment, as well as adjacent to microvessels Corresponding Author: Christine A. King, Department of Microbiology and where they monitor both blood and lymph circulation (5). As Immunology, SUNY Upstate Medical University, 750 East Adams St, Syracuse, immune surveillance cells (6), they have essential roles in both NY 13210. Phone: 315-464-5465; Fax: 315-464-4417; E-mail: [email protected] acute and chronic inflammation (5, 7). Given the shared ontog- doi: 10.1158/1078-0432.CCR-18-0873 eny of ECs and MCs, as well as their close association within all Ó2018 American Association for Cancer Research. vascularized tissues, it is not surprising that these cell types www.aacrjournals.org 5085 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst July 3, 2018; DOI: 10.1158/1078-0432.CCR-18-0873 Ayers et al. Translational Relevance Methods Cell culture Inflammation is a key driver of oncogenesis and modu- BCBL-1 PEL cells were maintained in RPMI medium 1640 lation of inflammation in the tumor microenvironment is containing 10% FBS, 1% penicillin–streptomycin, and emerging as a viable therapeutic approach. Kaposi sarcoma 0.05 mol/L b-mercaptoethanol. LAD2 (a kind gift from Arnold (KS), one of the most commonly diagnosed and aggressive Kirshenbaum, NIH, Bethesda, MD) and LUVA (a kind gift from cancers in sub-Saharan Africa, is an unusual cancer, depen- John Steinke, University of Virginia, Charlottesville, VA) MCs dent on infection with KS herpes virus (KSHV) and on were cultured in serum-free media (StemPro-34 SFM, Life Tech- inflammatory mediators. Despite extensive study the key nologies). HMC-1 clone 5C6 MCs were cultured in Iscove medi- cellular players and paracrine inflammatory mediators um (Gibco) and authenticated by the University of Arizona driving the disease are incompletely understood. Here genetics core by Promega PowerPlex assay and short tandem we identify a previously unrecognized role for potent repeat results compared with genomic databases. Because LAD2 proinflammatory mast cells (MCs) in KS. MCs are localized, and LUVA cell sequences are not currently available, these cells extensively activated, and infected by KSHV in KS tumors were authenticated by flow cytometry using MC-specific receptor and their clinical importance is underscored by the success- staining, CD117 (Thermo Fisher Scientific, clone 104D2) and ful treatment of a case of AIDS-KS with anti-MC therapy. FceRI (Thermo Fisher Scientific, clone AER-37). MC media were These data strongly suggest a specific role for MCs in supplemented with 2 mmol/L L-glutamine, 100 U/mL penicillin, KSHV-driven oncogenesis and identify MC antagonists 50 mg/mL streptomycin. A total of 100 ng/mL SCF was added to as a promising novel, pathogenesis-targeted therapeutic LAD2 cultures. LAD2 cells were fed by hemidepletion of media approach to KS. Our work adds to the growing body of twice a week. Primary human umbilical vein endothelial knowledge on the role of MCs in oncogenesis, and demon- (HUVEC) cells were purchased from Lonza. Cultures were strates the efficacy of using widely available MC-directed expanded in EBM-2 media (Lonza) supplemented with the therapies for treatment of KS. EGM-2 bullet kit in 6-well tissue culture plates coated with 0.1 % (w/v) gelatin in PBS and used between passage 2 and 5 for experiments. Cells were maintained at 37 Cin5%CO2. All cell lines were tested every 1 to 2 month(s) for Mycoplasma contam- cross-regulate each other. For example, chemical mediators ination using the MycoProbe Mycoplasma detection kit (R&D from MCs promote EC activation, which is required for the Systems). Cell lines were thawed and used for approximately regulated recruitment of leukocytes to inflamed tissues (8, 9). 2 months before being discarded and a fresh aliquot thawed for There is evidence that MC activation of ECs is key to the use in experiments with the exception of LAD2 cells that were pathophysiology of vascular compromise observed in severe cultured continuously as per instructions. dengue (10, 11) and influenza infections (12), and MCs are known to be permissible to infection by multiple viruses. fi Furthermore, there is accumulating evidence that MC Virus production and quanti cation activation is a promising target for adjunctive therapy in mul- KSHV was obtained from cultures of BCBL-1 cells that harbor  5 tiple cancer types. MCs are found in a wide range of malig- latent KSHV. Lytic reactivation of 5 10 BCBL-1 cells/mL was nancies, often associated with poor prognosis, increased metas- induced by addition